Method for preparing agonist for improving boar sperm motility

Abstract

A method for preparing an agonist for improving boar sperm motility includes: (1) collecting and infiltrating a testicular tissue of a young boar 3-5 days after born; (2) washing the tissue with PBS; (3) incubating the tissue in a cell culture medium, and centrifuging the cell suspension to remove a supernatant; (4) adding hyaluronidase and collagenase IV followed by shaking, and adding the cell culture medium and centrifuging to remove a supernatant; (5) adding trypsin and deoxyribonuclease followed by shaking, and adding the cell culture medium; (6) filtering the cell suspension by a cell sieve; (7) centrifuging to remove a supernatant, and adding the cell culture medium; (8) culturing and passaging the cells; (9) culturing and storing the cell suspension. The invention has the advantages of good application effect, low cost and simple production process.

What is claimed is: 
     
         1 . A method for preparing an agonist for improving boar sperm motility, comprising:
 (1) collecting a testicular tissue from a young boar 3 days after born, and infiltrating the testicular tissue in normal saline containing 1% of a double-antibody at 37° C. for 10-30 min;   (2) washing the infiltrated testicular tissue three times with PBS containing 1% of the double-antibody;   (3) incubating the testicular tissue in DMEM/F12 media at 37° C. for 30 min to fully disperse the testicular tissue; and transferring the dispersed testicular tissue to a centrifuge tube followed by centrifugation to remove a supernatant;   (4) adding 1.5 mL of hyaluronidase and 1.5 mL of collagenase IV to the centrifuge tube; shaking the centrifuge tube violently at 37° C. every other 2 min; after continuously shaken 2-3 times, adding 3 mL of DMEM/F12 media; and centrifuging the centrifuge tube to remove a supernatant;   (5) adding 1.5 mL of trypsin and 1.5 mL of deoxyribonuclease to the centrifuge tube; shaking the centrifuge tube violently at 37° C. every other 2 min; after continuously shaken 2-3 times, adding 3 mL of DMEM/F12 media to the centrifuge tube to produce a first cell suspension;   (6) filtering the first cell suspension sequentially by 80-mesh, 200-mesh and 300-mesh cell sieves;   (7) centrifuging the filtered first cell suspension to remove a supernatant; adding 4-6 mL of a cell culture medium to form a second cell suspension; mixing the second cell suspension by blowing; transferring the second cell suspension to a cell culture dish to culture for 2 h; and replacing the cell culture medium;   (8) passaging the cells cultured in the cell culture dish; and inoculating 1×10 6  cells to each of cell culture dishes having a diameter of 6 cm; and   (9) taking out the cell suspensions in the cell culture dishes after cultured for 8 h, and cryopreserving the cell suspensions at −20° C.   
     
     
         2 . The method of  claim 1 , wherein step (1) comprises:
 anesthetizing and castrating the young boar to collect testicles;   removing blood and adipose tissues with a pair of surgical scissors and cutting up the testicles;   adding the cut testicular tissue to the normal saline containing 1% of the double-antibody; and   culturing the testicular tissue at 37° C. and 5% CO 2  in an incubator for 10-30 min.   
     
     
         3 . The method of  claim 2 , wherein the young boar is collected 3-5 days after born. 
     
     
         4 . The method of  claim 1 , wherein the double-antibody is a mixture of penicillin (10000 IU) and streptomycin (10000 μg/mL). 
     
     
         5 . The method of  claim 1 , wherein the DMEM/F12 media is prepared by mixing F12 medium and DMEM in a ratio of 1:1 for clone culture at a density. 
     
     
         6 . The method of  claim 1 , wherein in step (7), the cell culture medium is a RPMI-1640 medium. 
     
     
         7 . The method of  claim 1 , wherein the cells are inoculated at a concentration of 1×10 6  cells/per cell culture dish of 6 cm in diameter.